Podoplanin is incorporated into virions released from 293T cells, and incorporation is essential for efficient CLEC-2-dependent HIV transmission. (A) 293T cells were transduced with retroviral vectors encoding EGFP and either a podoplanin-specific or a non-sense shRNA. Transduced cells were puromycin-selected and podoplanin (left panel) and EGFP expression (right panel) was determined by flow cytometry (using antibody 18H5). The average ± SEM of five independent experiments, for which GMCF was determined, is presented. Podoplanin expression on cells expressing control shRNA was set as 100%. (B) An env-defective NL4-3 proviral genome was transiently expressed in 293T cells transduced with vector encoding either podoplanin-specific shRNA or non-sense shRNA; the supernatants were harvested, and either processed directly or concentrated by size-exclusion filtration. Subsequently, the supernatants were analyzed for podoplanin and p24-content by Western blot. (C) The cells described in (A) were transfected with HIV-1 NL4-3 proviral DNA; the supernatants were harvested and their p24-content determined. Equal volumes of virus stocks containing 10 ng of p24-antigen were then incubated with the indicated B-THP cell lines and bound viruses transmitted to CEM×174 R5 targets. In parallel, direct infection of targets was assessed. The results represent the average ± SEM of six independent experiments carried out in triplicates with two independent virus stocks. Transmission of HIV-1 produced in 293T cells not transduced with shRNA-encoding vector was set as 100%. Control indicates B-THP cells stably transduced with empty vector. (D) The experiment was conducted as described in (C). However, HIV-1 transmission by platelets was examined. The results represent the average ± SEM of five independent experiments carried out in triplicates. The same virus stocks as in (C) were used. Mock indicates viruses produced in 293T cells not transduced with shRNA-encoding vector. GMCF: geometric mean channel fluorescence, ns shRNA: none-sense shRNA, PDPN: podoplanin, SEM: standard error of the mean.